Jan Domin

Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans

Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3‐kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3‐kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3‐phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3‐kinase. Here the molecular characterization of a new human PtdIns 3‐kinase with extensive sequence homology to Vps34p is described. PtdIns 3‐kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3‐kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

The Class II Phosphoinositide 3-Kinase C2α Is Activated by Clathrin and Regulates Clathrin-Mediated Membrane Trafficking

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.

Characterization of p150, an Adaptor Protein for the Human Phosphatidylinositol (PtdIns) 3-Kinase SUBSTRATE PRESENTATION BY PHOSPHATIDYLINOSITOL TRANSFER PROTEIN TO THE p150·;PtdIns 3-KINASE COMPLEX

Genetic and biochemical studies have shown that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is required for the efficient sorting and delivery of proteins to the vacuole. A human homologue of the yeast VPS34 gene product has recently been characterized as part of a complex with a cellular protein of 150 kDa (Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J., Domin, J., Panaretou, C., and Waterfield, M. D. (1995) EMBO J. 14, 3339-3348). Here, cDNA cloning is used to show that the amino acid sequence of this protein, termed p150, is 29.6% identical and 53% similar to the yeast Vps15p protein, an established regulator of Vps34p. Northern blot analysis showed a ubiquitous tissue distribution for p150 similar to that previously observed with PtdIns 3-kinase. Recombinant p150 associated with PtdIns 3-kinase in vitro in a stable manner, resulting in a 2-fold increase in lipid kinase activity. Addition of phosphatidylinositol transfer protein (PI-TP) further stimulated the lipid kinase activity of the p150·;PtdIns 3-kinase complex 3-fold. A PtdIns 3-kinase activity could also be co-immunoprecipitated from human cell lysates using anti-PI-TP antisera. This observation demonstrates that an interaction between a PtdIns 3-kinase and PI-TP occurs in vivo, which further implicates lipid transfer proteins in the regulation of PtdIns 3-kinase activity. These results suggest that the Vps15p·;Vps34p complex has been conserved from yeast to man and in both species is involved in protein trafficking.

The CC Chemokine Monocyte Chemotactic Peptide-1 Activates both the Class I p85/p110 Phosphatidylinositol 3-Kinase and the Class II PI3K-C2α

The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 ± 1.88 nm,n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 ± 4.2 nm, n= 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2α) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2α activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2α rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2α. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2α enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.

Class II Phosphoinositide 3-Kinases Are Downstream Targets of Activated Polypeptide Growth Factor Receptors

ABSTRACT The class II phosphoinositide 3-kinases (PI3K) PI3K-C2α and PI3K-C2β are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2α and PI3K-C2β represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2α and PI3K-C2β immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca2+ as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2α is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2β was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2β with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2β occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2β was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3′ phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2–3-fold increase in the activity of PI 3-kinase C2α (PI3K-C2α) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2α was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2α by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2α to associate with IRS-1 or insulin receptor. PI3K-C2α existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2α. The finding that insulin activates PI3K-C2α in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2α is a novel component of insulin-stimulated signaling cascades.

A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction

BACKGROUND Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate. Additionally, a PI 3-kinase activity which generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast. RESULTS We have identified a family of three distinct PI 3-kinases in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases. One of these family members, PI3K_92D, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase which is widely expressed throughout the Drosophila life cycle. The PI3K_68D cDNA encodes a protein of 210 kDa, which lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain. Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110. CONCLUSIONS A family of PI 3-kinases in Drosophila, including a novel class represented by PI3K_68D, is described. PI3K_68D has the potential to bind to signalling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity, so it could define a novel signal transduction pathway.

Neuromedin U--a study of its distribution in the rat.

The distribution of neuromedin U, a novel peptide originally isolated from porcine spinal cord, was investigated in the rat using a recently developed radioimmunoassay. High concentrations of neuromedin U-like immunoreactivity were found in the pituitary gland and gastrointestinal tract. Significant concentrations of immunoreactivity were also found in several regions of the rat brain, spinal cord and both male and female genitourinary tracts. In the small intestine, neuromedin U-like immunoreactivity was restricted to the submucosal muscular layers, suggesting localization in neurones rather than in epithelial cells. Chromatographic analysis of pituitary, spinal cord and gut revealed a single peak of immunoreactivity which did not co-elute with either synthetic porcine neuromedin U-25 nor neuromedin U-8, indicating inter-species molecular heterogeneity.

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.